Clinical and microbiological characteristics of bloodstream infection caused by Klebsiella pneumoniae harboring rmpA in Japanese adults

We investigated the clinical features of bloodstream infections (BSIs) caused by Klebsiella pneumoniae harboring rmpA and molecular characteristics of the bacteria. We retrospectively investigated adult patients with K. pneumoniae BSI from January 2010 to March 2021 at Nagasaki University Hospital. A matched case–control study in a 1:3 ratio was conducted to clarify the clinical and bacterial characteristics of BSI caused by rmpA-positive K. pneumoniae compared with those caused by rmpA-negative isolates. Antimicrobial susceptibility testing and multilocus sequence typing (MLST) were performed for rmpA-positive isolates. The rmpA was detected in 36 (13.4%) of the 268 isolates. Of these 36 isolates, 31 (86.1%) harbored iucA and 35 (97.2%) each possessed peg-344 and iroB; capsular types were identified as K1 in 9 (25.0%) and K2 in 10 isolates (27.8%). Contrarily, of the 108 rmpA-negative isolates, which were matched for case–control studies, 5 isolates (4.6%) harbored iucA and 1 (0.9%) each possessed peg-344 and iroB; 2 (1.9%) and 3 isolates (2.8%) had K1 and K2 capsular types, respectively. Among the rmpA-positive isolates, ST23/K1 (eight isolates) was the most frequent, followed by ST412/non-K1/K2 (seven isolates), ST86/K2 (five isolates), and ST268/non-K1/K2 (four isolates). In a multivariate analysis using clinical factors, liver abscess positively correlated with rmpA-positive isolates, whereas biliary tract infection and use of anticancer drugs negatively correlated with rmpA-positive isolates in patients with K. pneumoniae BSI. Considering the correlation between rmpA-positive isolates and clinical features, rmpA can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.

www.nature.com/scientificreports/ reduces colony mucoviscosity 10,11 , virulence in mice, and resistance to human serum 10 . Additionally, plasmidborne rmpA has been found to be an accurate marker of hvKp with high sensitivity (0.98) and specificity (0.93) 4 . Therefore, in this study, we focused on K. pneumoniae harboring plasmid-borne rmpA and investigated the molecular epidemiology and clinical features of BSI caused by the bacterium in our university hospital located in western Japan.

Materials and methods
Study design. We retrospectively investigated K. pneumoniae isolated from blood samples at Nagasaki University Hospital from January 2010 to March 2021. Adult patients aged 20 years or older, from whose blood samples, K. pneumoniae was isolated, were listed from our clinical laboratory database. The first isolate was selected when the bacteria were repeatedly isolated from individual patients during the study 12 . Of the isolates listed, those that were available were included in this study. We collected clinical and microbiological information obtained through routine practice from the medical records and laboratory systems in our hospital. Among patients for whom the isolates were available, a matched case-control study in a 1:3 ratio was conducted to clarify the clinical features of BSI caused by rmpA-positive K. pneumoniae and the characteristics of the bacterium. Cases and controls were defined as patients from whom rmpA-positive and rmpA-negative K. pneumoniae were isolated, respectively. For case-control matching, age (± 5 years) and sex-matched patients to each case were listed among patients from whom rmpA-negative K. pneumoniae was isolated, and three patients per case were randomly selected as controls using Microsoft Excel (Microsoft Corporation Microbiological analysis. Hypermucoviscosity was assessed using the string test, which was considered positive if the viscous string was greater than 5 mm in length when the colony was stretched using a loop on an agar plate 15 . Bacterial DNA was extracted using the boiling method previously described 12 , with minor modifications. Three to five colonies were mixed with 100 µL Tris-EDTA buffer containing 250 U/mL achromopeptidase (Wako Pure Chemical Industries, Ltd.). After incubation at 40 °C for 15 min, 250 µL of 10% Chelex 100 Resin (Bio-Rad) was added, and the mixture was boiled at 99 °C for 5 min, cooled on ice for 1 min, and centrifuged at 12,000 rpm for 1 min. The supernatant was used for the subsequent analyses.
In the multivariate analysis, variables with P < 0.2 in the univariate analysis were selected and adjusted using the conditional logistic regression model. Data were analyzed using JMP v16 (SAS Institute Inc.), and results with P < 0.05 were considered statistically significant.
Clinical features of BSI caused by K. pneumoniae harboring rmpA. We investigated the baseline characteristics and clinical features of BSI caused by rmpA-positive K. pneumoniae, compared with those caused by rmpA-negative isolates (Table 4). Of the 144 patients analyzed, 91 (63.2%) developed K. pneumoniae BSI in the hospital, and the rates were similar between the rmpA-positive and rmpA-negative groups (63.9% and 63.0%, respectively). The use of anticancer drugs was significantly higher in the rmpA-negative group than in the rmpApositive group. Similarly, the presence of malignancy tended to be higher in the rmpA-negative group than in  www.nature.com/scientificreports/ the rmpA-positive group, but the difference was not significant. Other comorbidities and use of medical devices did not differ between the groups. The biliary tract was the most frequent infection site with BSI (30 patients, 27.8%) in the rmpA-negative group, and the rate was higher than that in the rmpA-positive group (three patients, 8.3%). Conversely, liver abscess was a more frequent infection in the rmpA-positive group (eight patients, 22.2%) than in the rmpA-negative group (eight patients, 7.4%). Disease severity assessed using the Pitt bacteremia score was similar, and the mortality rates did not show significant differences between the groups.
Conditional regression analysis was performed to evaluate the correlation between rmpA-positive isolates and the clinical factors of patients with K. pneumoniae BSI. Variables with P < 0.2 in the univariate analysis (Table 4) were used for the analysis. The presence of liver abscess positively correlated with rmpA-positive isolates, whereas biliary tract infection and the use of anticancer drugs showed a negative correlation with rmpA-positive isolates in patients with K. pneumoniae BSI (Table 5).

Discussion
Our study demonstrated the molecular epidemiology of K. pneumoniae harboring rmpA and the clinical features of BSI caused by the bacterium in our university hospital. Of the 268 K. pneumoniae isolates from blood, rmpA was detected in 13.4%. After case-control matching (rmpA-positive, 36 isolates; rmpA-negative, 108 isolates), the positive rates of iucA, peg-344, and iroB were remarkably higher in the rmpA-positive group (86.1%, 97.2%, and 97.2%, respectively) than in the rmpA-negative group (4.6%, 0.9%, and 0.9%, respectively). In addition to rmpA, iucA, peg-344, and iroB have been reported to be accurate markers of hvKp 4 . The high detection rates of these markers in rmpA-positive isolates support that rmpA is a useful marker of hvKp. Furthermore, K1 and K2 capsular types were identified in 25.0% and 27.8% of the isolates, respectively, in the rmpA-positive group, which were clearly higher than those in the rmpA-negative group (1.9% and 2.8%, respectively). The STs of K. pneumoniae from patients with BSI vary geographically 20 . Our results showed that ST23/K1 was the most prevalent (eight of 36 isolates) in rmpA-positive K. pneumoniae causing BSI, which is supported by the findings of a previous study on hvKp from Japan 21 . Additionally, we identified ST65/K2 and ST86/K2, similar to that in a previous study in Japan 21 . This study showed the clinical characteristics of BSI caused by K. pneumoniae harboring rmpA. Liver abscess was recorded in 22.2% of the patients with BSI in the rmpA-positive group, three times more frequently than that Table 4. Clinical characteristics of bloodstream infections caused by rmpA-positive and rmpA-negative K. pneumoniae. Data are expressed as median (interquartile range) or number (%). a Clinical diagnosis with K. pneumoniae isolation from each site. b Clinical diagnosis regardless of K. pneumoniae isolation from each site. c Infectious hepatic cysts in two patients were included.   22 . Furthermore, ST23/K1 (three isolates) and ST65/K2 (two isolates) were identified in five (62.5%) of the eight rmpA-positive K. pneumoniae isolates that caused liver abscess in our study, which is consistent with previous reports that they are the common ST/capsular types associated with liver abscess in East Asian countries [23][24][25] . The remaining types that caused liver abscess were ST412/non-K1/K2 (two isolates) and ST268/non-K1/K2 (one isolate). Meanwhile, the use of anticancer drugs and the presence of biliary tract infection negatively correlated with rmpA-positive isolates. Classical K. pneumoniae is known to cause bacteremia especially in immunocompromised patients 2 . Therefore, the use of anticancer drugs may reflect the immunocompromised condition of the host. Additionally, biliary tract was a frequent infection site in the rmpA-negative group (27.8%) compared with that in the rmpA-positive group (8.3%) in this study. A recent study reported a similar result that biliary tract infection was observed more frequently in classical K. pneumoniae BSI 26 .
This study has a few limitations. First, as this was a retrospective study, some variables of clinical factors might not have been recorded by attending physicians. Second, the sample size was limited because this study was conducted in a single center, and some isolates were unavailable during the study period. Finally, because we focused on rmpA-positive isolates in this study, we could not analyze the microbiological characteristics of rmpA-negative isolates in detail.
In conclusion, our study revealed the molecular epidemiology of K. pneumoniae harboring rmpA, isolated from patients with BSI in our hospital. The presence of rmpA correlated with the clinical characteristics of K. pneumoniae BSI and can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.

Data availability
The MLST allele sequences are available in Nagasaki University's Academic Output Site (http:// hdl. handle. net/ 10069/ 00041 907), and the allele numbers are provided in Supplementary Table 1.